MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

In Vivo Genome-Wide PGR Binding in Pregnant Human Myometrium Identifies Potential Regulators of Labor.

The alterations in myometrial biology during labor are not well understood. The myometrium is the contractile portion of the uterus and contributes to labor, a process that may be regulated by the steroid hormone progesterone. Thus, human myometrial tissues from term pregnant in-active-labor (TIL) and term pregnant not-in-labor (TNIL) subjects were used for genome-wide analyses to elucidate potential future preventive or therapeutic targets involved in the regulation of labor. Using myometrial tissues directly subjected to RNA sequencing (RNA-seq), progesterone receptor (PGR) chromatin immunoprecipitation sequencing (ChIP-seq), and histone modification ChIP-seq, we profiled genome-wide changes associated with gene expression in myometrial smooth muscle tissue in vivo. In TIL myometrium, PGR predominantly occupied promoter regions, including the classical progesterone response element, whereas it bound mainly to intergenic regions in TNIL myometrial tissue. Differential binding analysis uncovered over 1700 differential PGR-bound sites between TIL and TNIL, with 1361 sites gained and 428 lost in labor. Functional analysis identified multiple pathways involved in cAMP-mediated signaling enriched in labor. A three-way integration of the data for ChIP-seq, RNA-seq, and active histone marks uncovered the following genes associated with PGR binding, transcriptional activation, and altered mRNA levels: ATP11A, CBX7, and TNS1. In vitro studies showed that ATP11A, CBX7, and TNS1 are progesterone responsive. We speculate that these genes may contribute to the contractile phenotype of the myometrium during various stages of labor. In conclusion, we provide novel labor-associated genome-wide events and PGR-target genes that can serve as targets for future mechanistic studies.

Progesterone receptor-DNA methylation crosstalk regulates depletion of uterine leiomyoma stem cells: A potential therapeutic target.

Uterine leiomyoma (LM) is the most common tumor in women. Via its receptor (PGR) expressed in differentiated LM cells, progesterone stimulates paracrine signaling that induces proliferation of PGR-deficient LM stem cells (LSCs). Antiprogestins shrink LM but tumors regrow after treatment cessation possibly due to persisting LSCs. Using sorted primary LM cell populations, we found that the PGR gene locus and its target cistrome are hypermethylated in LSCs, inhibiting the expression of genes critical for progesterone-induced LSC differentiation. PGR knockdown shifted the transcriptome of total LM cells toward LSCs and increased global DNA methylation by regulating TET methylcytosine dioxygenases. DNA methylation inhibitor 5'-Aza activated PGR signaling, stimulated LSC differentiation, and synergized with antiprogestin to reduce tumor size in vivo. Taken together, targeting the feedback loop between DNA methylation and progesterone signaling may accelerate the depletion of LSCs through rapid differentiation and sensitize LM to antiprogestin therapy, thus preventing tumor regrowth.

Activation of protein kinase B by WNT4 as a regulator of uterine leiomyoma stem cell function.

OBJECTIVE: To investigate the functional interaction between the Wnt/ß-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). DESIGN: Laboratory study. SETTING: Research laboratory. PATIENT(S): Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression, protein phosphorylation, and cell proliferation. RESULT(S): Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/ß-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/ß-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated ß-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. CONCLUSION(S): Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent ß-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell-enriched Population by Stimulating Their Differentiation.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell-like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell-like cells harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed the genes important for myometrium- and LM-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5'-Aza, stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of LM tumors.